To identify genes involved in the regulation of plasma high density lipoprotein (HDL) cholesterol (HDL-C) levels, patients with low HDL-C and age-and sex-matched controls (normal HDL-C) were extensively characterized. Comparative transcriptome analysis was carried out in cholesterol-loaded monocytederived macrophages from low HDL subjects segregated into groups with or without cholesterol efflux defects or ABCA1 mutations. Clusters of differentially regulated genes were evident in the low HDL groups as compared with controls. Of particular note, expression of cathepsin D (CTSD), a lysosomal proteinase, was reduced by ϳ50% in monocyte-derived macrophages of low HDL-C subjects, most significantly those with cholesterol efflux defects but without mutations in ABCA1 (p < 0.01). These results were verified by reverse transcription-PCR and replicated in a second cohort. We show here that blocking the activity or expression of CTSD, by pepstatin or CTSD small interfering RNA, respectively, reduced ABCA1 expression and protein abundance in both macrophages and CHO cells and apolipoprotein A-I-mediated lipid efflux by more than 70%. Conversely, expression of CTSD increased both ABCA1 mRNA expression and cellular ABCA1 protein. Consistent with its role in the proteolytic processing of prosaposin, inactivation of CTSD function resulted in the accumulation of glycosphingolipid and free cholesterol in late endosomes/lysosomes, a phenotype similar to NPC1 deficiency. Inhibition of CTSD also caused retention of ABCA1 in lysosomal compartments, reducing its trafficking to the plasma membrane. These studies demonstrate a novel and potentially important role for CTSD in intracellular cholesterol trafficking and ABCA1-mediated efflux. Therefore, decreased CTSD expression may contribute to low plasma HDL-C levels.

Objective— To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice. Methods and Results— SR-BI deficient (−/−) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI −/− × CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI −/− mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI −/− × CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet ( P <0.003). Conclusions— Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

The scavenger receptor BI (SR-BI) is highly expressed in hepatocytes, where it mediates the uptake of lipoprotein cholesterol, promotes the secretion of cholesterol into bile, and protects against atherosclerosis. Despite a strong correlation between the hepatic expression of SR-BI and biliary cholesterol secretion, little is known about SR-BI trafficking in response to changes in sterol availability. Using a well characterized polarized hepatocyte cell model, WIF-B, we determine that in cholesterol-depleted cells, SR-BI is extensively located on the basolateral surface, where it can access circulating lipoproteins. However, in response to cholesterol loading, SR-BI undergoes a slow transcytosis to the apical bile canaliculus independently of lipoprotein binding and new protein synthesis. In cholesterolreplete WIF-B cells, SR-BI that resides on the canalicular membrane is dynamically associated with defined microdomains and does not rapidly recycle to and from the subapical or basolateral regions. Taken together, these data demonstrate that hepatic SR-BI transcytosis is regulated by cholesterol and suggest that SR-BI has a stationary function on the bile canaliculus. High density lipoproteins (HDL)2 have a functional role in the protection against cardiovascular disease. HDL mediates both cholesterol removal from lipid-laden macrophages and delivery to the liver for subsequent biliary secretion in a process termed "macrophage reverse cholesterol transport." The cholesterol removal is mediated, in part, by the well established HDL receptor, scavenger receptor class BI (SR-BI) (1) (reviewed in Ref. 2).SR-BI is a two-transmembrane domain cell surface glycoprotein with short intracellular N-and C-terminal domains (3). The receptor is ubiquitously expressed at low levels and is highly expressed in steroidogenic, intestinal, and hepatic tissues (1). SR-BI binds a large array of ligands, including HDL and native or modified low density lipoproteins (LDL).Hepatic SR-BI protects against atherosclerosis by promoting the final stages of macrophage reverse cholesterol transport (5). In contrast to the holo-particle uptake of the LDL receptor pathway, SR-BI mediates cholesterol, cholesteryl ester, and phospholipid uptake via a selective pathway whereby lipids are transferred down their concentration gradient through a hydrophobic channel into the membrane (6). Importantly, if the concentration gradient is reversed, SR-BI can also perform cholesterol efflux (7).Although lipid delivery to and from lipoprotein particles occurs mainly on the plasma membrane, both SR-BI and HDL have been shown to internalize into and recycle from endocytic compartments (8 -10). SR-BI also localizes on the apical domain in gall bladder epithelial cells (11), testicular Sertoli cells (12), isolated primary mouse hepatocytes (13), primary mouse hepatocyte couplets (8), and hepatic tissues sections (14) and has been shown to undergo regulated transcytotic movement in polarized Madin-Darby canine kidney cells (15).Our laboratory has recently demonstra...

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